PROJECT SUMMARY Duchenne muscular dystrophy (DMD) affects 1 in 5000 live male births making it the most common form of muscular dystrophy worldwide. Patients with this X-linked, progressive neuromuscular disorder develop progressive muscle loss typically accompanied by cardiac arrhythmias, respiratory complications, and a loss of ambulation by their teen years. DMD is caused by inactivating mutations in the DYSTROPHIN (DMD) gene, ultimately resulting in the breakdown of muscle cell membranes and myofiber death. No cure exists and current therapeutic treatments focus on the ability to slow muscle wasting with corticosteroids or the alleviation of secondary comorbidities. Recently, it has been demonstrated that specific muscle-enriched microRNAs play important roles in DMD muscle formation, regeneration, and disease progression. Our laboratory has previously shown that muscle-specific miR-486 transgenic overexpression can block dystrophic pathology and prevent muscle loss in a DMD mouse model. The mechanistic role of miR-486 in DMD pathology has yet to be investigated. The proposed aims of this fellowship focus on elucidating the influence of miR-486 on gene expression in skeletal muscle pathology in DMD. Using molecular, cellular, in vivo, and ex vivo techniques, I will test my overarching hypothesis that miR-486 is a significant disease modifier of DMD whose downregulation contributes to the progression of dystrophic pathology. Specific Aim 1 ? I have learned how to assess global physical function in mice, analyze in vivo cardiac function using echocardiography, tissue and organ dissection, tissue sectioning, histochemistry/immunofluorescent staining and imaging, and RNA-seq analysis for microRNA target prediction and pathway analyses. Specific Aim 2 ? I will test the hypothesis that mRNA targets of miR-486 are upregulated in mdx5cv mice and that corresponding mRNA upregulation is contributing to an exacerbation of dystrophic disease progression at pre-symptomatic (5-8 weeks), symptomatic (4-6 months), and severe disease (10-16 months) cohorts. 2A: I will characterize histology and physiological function of miR-486 KO mice using standard locomotor, histochemical, and immunofluorescent assays for skeletal muscle. 2B: Because of the known role of miR-486 in muscle stem cell differentiation, I will induce skeletal muscle injury in miR-486 KO mice via cardiotoxin and assess for capacity for regeneration via ex vivo cell culture and whole muscle histological chemical and immunofluorescent staining. 2C: I will perform in silico analysis on previously-completed RNA-sequencing of miR-486 KO mouse TA muscle compared to WT to identify potential mRNA targets and then perform cell culture validation assays to confirm direct biological targets of miR-486. 2D: I will validate overexpression of miR-486 targets in mdx5cv mouse muscle via qPCR, western blot, and immunofluorescent staining. Specific Aim 3 ? My project will contribute to the overarching goal of identifying underlying mechanisms of disease progression in DMD for future therapeutic modulation. Specifically, this project will reveal promising data regarding the mechanism by which miR-486 is contributing to DMD disease pathology. This is novel work contributing to the use of miR-486 expression as a biomarker for DMD disease progression that has potential for future clinical application. I will take this experience to a postdoctoral fellowship in an academic research lab that studies motor neuron diseases where I can continue pursue biomarker identification with the intent of pursuing therapeutic interventions and quality of life improvement for patients living with neuromuscular diseases.